Project Summary Listeria monocytogenes is a facultative intracellular pathogen that escapes from the vacuole and takes residence in the host cytosol. During infection, the transcription factor PrfA is responsible for recognizing that L. monocytogenes is present in the host cytosol and for activating the suite of virulence factors required for pathogenesis. The cytosolic environmental niche is defined by a unique hybrid aerobic + reduced condition and recent studies suggest that this combination of properties might represent the cue that L. monocytogenes senses to interpret an intracellular localization. To follow up on this finding, a preliminary transposon library screen has been conducted to identify L. monocytogenes genes important for the sensing of reducing properties. From this screen, an 8-gene locus that?s conserved in over 200 gram-positive bacteria was identified. Preliminary experiments demonstrate that these genes encode an abundantly expressed extracellular electron transport chain (EETC) that links extra and intracellular electron pools. Experiments proposed here seek to delineate the role of this novel EETC in L. monocytogenes host sensing and to determine additional functional roles in central metabolism and bacterial competition.